The Synergistic Effect Of AZT Combined With Arsenic Trioxide Against The HepG2 Cell

Objective: To investigate the effects of arsenic trioxide(As2O3) combined with 3’-azido-3’-deoxythymidine(AZT) on proliferative inhibition and apoptosis induction of hepatoma Hep G2 cells and related role of Egr-1 gene, in support of clinical treatment against hepatoma.Methods: Human hepatocelluar carcinoma Hep G2 cells were cultured in vitro. MTT assay was used to examine the effect of different concentration of As2O3, AZT and their combinations on the proliferation of the Hep G2 cells, the combination indexes(CI) of As2O3 and AZT were calculated on Calcusyn 2.0 software. The apoptotic rates of Hep G2 cells after treatment with As2O3, AZT and their combinations were detected by flow cytometry(FCM). The expression levels of Caspase-3, Bcl-2 and Bax m RNAs and proteins were analyzed through RT-PCR and Western blotting, respectively.Hep G2 cells were electrotransfected with Egr-1 si RNA. They were divided as blank control(Hep G2), nonspecific control(Hep G2/NC) and Egr-1 si RNA group(Hep G2/si RNA). After the cells were treated with the combination of As2O3(2 μmol/L) and AZT(20 μmol/L), the transfection efficiency, the apoptosis rates and proliferation inhibition rates of cells in 3 groups were detected with FCM and MTT method,respectively. The expression levels of Egr-1 m RNA and protein were analyzed through q-PCR and Western blotting, respectively.Result: Proliferation inhibition effects of Hep G2 cells treated with As2O3, AZT and the combination all showed dose-time dependence. Although, the inhibitory effect of As2O3 or AZT alone was not obvious, the proliferation inhibitory rates of cells treated with combination of As2O3 and AZT with a fixed AZT(10μmol/L, 20 μmol/L) concentration and different As2O3 concentrations were all significantly higher than those by As2O3 or AZT alone with same concentrations. There were statistically significant between them(P<0.05).Calculated on Calcu Syn 2.0 statistical software, the CI values of the combination of As2O3 and AZT with a fixed final AZT concentration(10, 20 μmol/L) and different final As2O3 concentrations(final concentration<8 μmol/L) on Hep G2 cells treated for 48, 72 and 96 hours were less than 1 except the combination of AZT with a concentrations of As2O3(final concentration>8 μmol/L) for 24 hours, which point synergistic effect between AZT and a low concentrations of As2O3 on proliferation inhibition of Hep G2 cells.The apoptotic rate of Hep G2 cells after treatment with the combination of As2O3 and AZT was higher than those of the control cells(without any treatment) and As2O3 and AZT alone groups(P<0.05), which indicate synergistic effect between As2O3 and a low concentrations of AZT on apoptosis induction of Hep G2 cells.The expression levels of Caspase-3 and Bax m RNAs and proteins in combination of As2O3 and AZT group were higher than those of the control group(without any treatment) and As2O3 or AZT alone groups(P<0.05). The expression levels of Bcl-2 m RNA and protein in combination of As2O3 and AZT group were lower than those of As2O3 or AZT alone groups(P<0.05). The ratios of Bax/Bcl-2 m RNA and protein in combination of As2O3 and AZT group were higher than those of the control group(P<0.05).Transfection efficiency was 55.9% after transient transfection. Compared with nonspecific control group and blank control group, Egr-1 si RNA group cell proliferation was increased and the expression level of Egr-1 m RNA was significantly decreased. The value of combination indexes were less than 1 after treatment with As2O3 and AZT on blank control group and nonspecific control group, indicating obvious synergistic effect between them. There were no significant difference between As2O3 or AZT alone and their combination on Egr-1 si RNA group. The value of combination indexes was more than 1. The apoptosis rate of Egr-1 si RNA group was lower compared with blank control group(P<0.01). The expression of Egr-1 m RNA and protein was up-regulated in both 3 groups after treated with 2 μmol/L As2O3 and 20 μmol/L AZT than control group, among them Egr-1 si RNA group was the highest and there were no difference between other 2 groups(P>0.05).Conclusion: As2O3 combined with AZT proved significant synergistic effect on Hep G2 cells in proliferation inhibition and apoptosis induction, which can have relation with up-regulation of Caspase-3 and Bax expressions, and down-regulation of Bcl-2 expression. There is a correlation between Egr-1 and synergistic effect on apoptosis induction and proliferation inhibition of Hep G2 cells.